Histophilus somni is a major infectious agent worldwide. This bacterium produces a large fibrillar surface antigen called IbpA (immunoglobulin binding protein A). Pasturella multocida, the causative agent of the most common bacterial infection due to an animal bite, also produces a large surface antigen known as PfhB2. PfhB2 shares extensive amino acid sequence identity with IbpA. This suggests that there is a small family of bacteria that harbors these 4000 amino acid toxins. We focused our attention on IbpA since convalescent serum from symptomatic animals infected with H. somni recognizes IbpA, while serum from asymptomatic animals does not. As such, the presence of IbpA directly correlates with H. somni virulence. The COOH-terminus of IbpA is homologous to the Yersinia type III effector protein, YopT, one of several virulence factors used by Yersinia to compromise the host immune system. We previously demonstrated that Yersinia YopT functions as a cysteine protease that cleaves and inactivates Rho GTPases. Our hypothesis was that IbpA's filamentous hemagglutinin-like domains mediate attachment to host cells, while its COOH-terminus containing the YopT homology sequence serves as a cytotoxic effector when internalized into host cells. Contrary to our expectations, we observed that the YopT-like domain of IbpA does not disrupt the actin cytoskeleton despite its conservation of the key catalytic C/H/D triad. Instead, we identified a virulence determinant within IbpA that is localized to a portion of the protein known as the Fic (filamentation induced by c-AMP) domain. Fic domains are found in approximately 1500 proteins encoded by bacteria and are present as single copy genes in many eukaryotic genomes. The function of these Fic domains is unknown. We demonstrated that the Fic domains of IbpA induce cytotoxicity by targeting the host GTPases, RhoA, Rac and Cdc42. The Fic domains of IbpA block signaling of these GTPases by using ATP to catalyze the covalent addition of adenosine monophosphate (AMP) to a tyrosine (Tyr) residue in the GTPase switch I region. This covalent AMP addition leads to a block in downstream signaling of the GTPases, which in turn results in cytotoxicity. The ability to add AMP to the GTPases is dependent on the presence of a conserved histidine (His) in the Fic domain's core motif, HPFxxGNGR. In summary, we have identified a new class of proteins that play an important novel role in bacterial pathogenesis. Our results also suggest that addition of AMP to host proteins may be an underappreciated post-translational modification in both prokaryotes and eukaryotes. The specific aims for this application are: (1) Determine if Fic domains from bacteria and higher eukaryotes all have adenylyl transferase activity. (2A) Study the kinetic properties and catalytic mechanisms of Fic-mediated adenylylation. (2B) Elucidate the cellular substrates of Fic domain containing enzymes. (3) Determine how IbpA enters mammalian cells. (4) Determine the X-ray structures of the IbpA's Fic domain as well as a Fic domain complexed with a non-hydrolyzed analogue of ATP. (5) Determine the X-ray structure of the protein complex containing a Fic domain, a non-hydrolyzed ATP analogue and RhoA. These studies will provide a detailed understanding of the structure and mechanism used by the Fic domain to carry out this novel post-translational modification. This will collectively advance our understanding of how the Fic domain containing proteins function in bacterial pathogenesis. PUBLIC HEALTH RELEVANCE: Prior to our work, the function of the Fic domain in bacterial pathogenesis was unknown despite its presence in over 1400 proteins from a wide variety of bacteria. We have demonstrated that the Fic domain of IbpA, a large toxin found in Histophilus somni, can disrupt the actin cytoskeleton by using ATP as a substrate to catalyze the addition of AMP to several host GTPases. This covalent addition of AMP blocks signal transduction pathways in the host that are important for combating bacterial infections.